Therapeutic CFTR Correction Normalizes Systemic and Lung-Specific S1P Level Alterations Associated with Heart Failure.

Heart failure (HF) is among the main causes of death worldwide. Alterations of sphingosine-1-phosphate (S1P) signaling have been linked to HF as well as to target organ damage that is often associated with HF. S1P's availability is controlled by the cystic fibrosis transmembrane regulator (CFTR), which acts as a critical bottleneck for intracellular S1P degradation. HF induces CFTR downregulation in cells, tissues and organs, including the lung. Whether CFTR alterations during HF also affect systemic and tissue-specific S1P concentrations has not been investigated. Here, we set out to study the relationship between S1P and CFTR expression in the HF lung. Mice with HF, induced by myocardial infarction, were treated with the CFTR corrector compound C18 starting ten weeks post-myocardial infarction for two consecutive weeks. CFTR expression, S1P concentrations, and immune cell frequencies were determined in vehicle- and C18-treated HF mice and sham controls using Western blotting, flow cytometry, mass spectrometry, and qPCR. HF led to decreased pulmonary CFTR expression, which was accompanied by elevated S1P concentrations and a pro-inflammatory state in the lungs. Systemically, HF associated with higher S1P plasma levels compared to sham-operated controls and presented with higher S1P receptor 1-positive immune cells in the spleen. CFTR correction with C18 attenuated the HF-associated alterations in pulmonary CFTR expression and, hence, led to lower pulmonary S1P levels, which was accompanied by reduced lung inflammation. Collectively, these data suggest an important role for the CFTR-S1P axis in HF-mediated systemic and pulmonary inflammation.

Endoscopic atomization of mesenchymal stromal cells: in vitro study for local cell therapy of the lungs.

BACKGROUND AIMS: Cell-based therapies of pulmonary diseases with mesenchymal stromal cells (MSCs) are increasingly under experimental investigation. In most of these, MSCs are administered intravenously or by direct intratracheal instillation. A parallel approach is to administer the cells into the lung by endoscopic atomization (spraying). In a previous study, the authors developed a flexible endoscopic atomization device that allows administration of respiratory epithelial cells in the lungs with high survival. METHODS: In this study, the authors evaluated the feasibility of spraying MSCs with two different endoscopic atomization devices (air and pressure atomization). Following atomization, cell viability was evaluated with live/dead staining. Subsequent effects on cytotoxicity, trilineage differentiation and expression of MSC-specific markers as well as on MSC metabolic activity and morphology were analyzed for up to 7 days. RESULTS: MSC viability immediately after spraying and subsequent metabolic activity for 7 days was not influenced by either of the devices. Slightly higher cytotoxicity rates could be observed for pressure-atomized compared with control and air-atomized MSCs over 7 days. Flow cytometry revealed no changes in characteristic MSC cell surface marker expression, and morphology remained unchanged. Standard differentiation into osteocytes, chondrocytes and adipocytes was inducible after atomization. CONCLUSIONS: In the literature, a minimal survival of 50% was previously defined as the cutoff value for successful cell atomization. This is easily met with both of the authors' devices, with more than 90% survival. Thus, there is a potential role for atomization in intrapulmonary MSC-based cell therapies, as it is a feasible and easily utilizable approach based on clinically available equipment.

Applications and Approaches for Three-Dimensional Precision-Cut Lung Slices. Disease Modeling and Drug Discovery.

Chronic lung diseases (CLDs), such as chronic obstructive pulmonary disease, interstitial lung disease, and lung cancer, are among the leading causes of morbidity globally and impose major health and financial burdens on patients and society. Effective treatments are scarce, and relevant human model systems to effectively study CLD pathomechanisms and thus discover and validate potential new targets and therapies are needed. Precision-cut lung slices (PCLS) from healthy and diseased human tissue represent one promising tool that can closely recapitulate the complexity of the lung's native environment, and recently, improved methodologies and accessibility to human tissue have led to an increased use of PCLS in CLD research. Here, we discuss approaches that use human PCLS to advance our understanding of CLD development, as well as drug discovery and validation for CLDs. PCLS enable investigators to study complex interactions among different cell types and the extracellular matrix in the native three-dimensional architecture of the lung. PCLS further allow for high-resolution (live) imaging of cellular functions in several dimensions. Importantly, PCLS can be derived from diseased lung tissue upon lung surgery or transplantation, thus allowing the study of CLDs in living human tissue. Moreover, CLDs can be modeled in PCLS derived from normal lung tissue to mimic the onset and progression of CLDs, complementing studies in end-stage diseased tissue. Altogether, PCLS are emerging as a remarkable tool to further bridge the gap between target identification and translation into clinical studies, and thus open novel avenues for future precision medicine approaches.

Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases 2017. An Official American Thoracic Society Workshop Report.

The University of Vermont Larner College of Medicine, in collaboration with the National Heart, Lung, and Blood Institute (NHLBI), the Alpha-1 Foundation, the American Thoracic Society, the Cystic Fibrosis Foundation, the European Respiratory Society, the International Society for Cell & Gene Therapy, and the Pulmonary Fibrosis Foundation, convened a workshop titled "Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases" from July 24 through 27, 2017, at the University of Vermont, Burlington, Vermont. The conference objectives were to review and discuss current understanding of the following topics: 1) stem and progenitor cell biology and the role that they play in endogenous repair or as cell therapies after lung injury, 2) the emerging role of extracellular vesicles as potential therapies, 3) ex vivo bioengineering of lung and airway tissue, and 4) progress in induced pluripotent stem cell protocols for deriving lung cell types and applications in disease modeling. All of these topics are research areas in which significant and exciting progress has been made over the past few years. In addition, issues surrounding the ethics and regulation of cell therapies worldwide were discussed, with a special emphasis on combating the growing problem of unproven cell interventions being administered to patients with lung diseases. Finally, future research directions were discussed, and opportunities for both basic and translational research were identified.

Lung bioengineering: advances and challenges in lung decellularization and recellularization.

PURPOSE OF REVIEW: Bioengineering the lung based on its natural extracellular matrix (ECM) offers novel opportunities to overcome the shortage of donors, to reduce chronic allograft rejections, and to improve the median survival rate of transplanted patients. During the last decade, lung tissue engineering has advanced rapidly to combine scaffolds, cells, and biologically active molecules into functional tissues to restore or improve the lung's main function, gas exchange. This review will inspect the current progress in lung bioengineering using decellularized and recellularized lung scaffolds and highlight future challenges in the field. RECENT FINDINGS: Lung decellularization and recellularization protocols have provided researchers with tools to progress toward functional lung tissue engineering. However, there is continuous evolution and refinement particularly for optimization of lung recellularization. These further the possibility of developing a transplantable bioartificial lung. SUMMARY: Bioengineering the lung using recellularized scaffolds could offer a curative option for patients with end-stage organ failure but its accomplishment remains unclear in the short-term. However, the state-of-the-art of techniques described in this review will increase our knowledge of the lung ECM and of chemical and mechanical cues which drive cell repopulation to improve the advances in lung regeneration and lung tissue engineering.

Cigarette smoke alters the secretome of lung epithelial cells.

Cigarette smoke is the most relevant risk factor for the development of lung cancer and chronic obstructive pulmonary disease. Many of its more than 4500 chemicals are highly reactive, thereby altering protein structure and function. Here, we used subcellular fractionation coupled to label-free quantitative MS to globally assess alterations in the proteome of different compartments of lung epithelial cells upon exposure to cigarette smoke extract. Proteomic profiling of the human alveolar derived cell line A549 revealed the most pronounced changes within the cellular secretome with preferential downregulation of proteins involved in wound healing and extracellular matrix organization. In particular, secretion of secreted protein acidic and rich in cysteine, a matricellular protein that functions in tissue response to injury, was consistently diminished by cigarette smoke extract in various pulmonary epithelial cell lines and primary cells of human and mouse origin as well as in mouse ex vivo lung tissue cultures. Our study reveals a previously unrecognized acute response of lung epithelial cells to cigarette smoke that includes altered secretion of proteins involved in extracellular matrix organization and wound healing. This may contribute to sustained alterations in tissue remodeling as observed in lung cancer and chronic obstructive pulmonary disease.

Preclinical validation and imaging of Wnt-induced repair in human 3D lung tissue cultures.

Chronic obstructive pulmonary disease (COPD) is characterised by a progressive loss of lung tissue. Inducing repair processes within the adult diseased lung is of major interest and Wnt/ß-catenin signalling represents a promising target for lung repair. However, the translation of novel therapeutic targets from model systems into clinical use remains a major challenge.We generated murine and patient-derived three-dimensional (3D) ex vivo lung tissue cultures (LTCs), which closely mimic the 3D lung microenvironment in vivo. Using two well-known glycogen synthase kinase-3ß inhibitors, lithium chloride (LiCl) and CHIR 99021 (CT), we determined Wnt/ß-catenin-driven lung repair processes in high spatiotemporal resolution using quantitative PCR, Western blotting, ELISA, (immuno)histological assessment, and four-dimensional confocal live tissue imaging.Viable 3D-LTCs exhibited preserved lung structure and function for up to 5 days. We demonstrate successful Wnt/ß-catenin signal activation in murine and patient-derived 3D-LTCs from COPD patients. Wnt/ß-catenin signalling led to increased alveolar epithelial cell marker expression, decreased matrix metalloproteinase-12 expression, as well as altered macrophage activity and elastin remodelling. Importantly, induction of surfactant protein C significantly correlated with disease stage (per cent predicted forced expiratory volume in 1 s) in patient-derived 3D-LTCs.Patient-derived 3D-LTCs represent a valuable tool to analyse potential targets and drugs for lung repair. Enhanced Wnt/ß-catenin signalling attenuated pathological features of patient-derived COPD 3D-LTCs.

Ivacaftor therapy post myocardial infarction augments systemic inflammation and evokes contrasting effects with respect to tissue inflammation in brain and lung.

Acquired cystic fibrosis transmembrane regulator (CFTR) dysfunctions have been associated with several conditions, including myocardial infarction (MI). Here, CFTR is downregulated in brain, heart, and lung tissue and associates with inflammation and degenerative processes. Therapeutically increasing CFTR expression attenuates these effects. Whether potentiating CFTR function yields similar beneficial effects post-MI is unknown. The CFTR potentiator ivacaftor is currently in clinical trials for treatment of acquired CFTR dysfunction associated with chronic obstructive pulmonary disease and chronic bronchitis. Thus, we tested ivacaftor as therapeutic strategy for MI-associated target tissue inflammation that is characterized by CFTR alterations. MI was induced in male C57Bl/6 mice by ligation of the left anterior descending coronary artery. Mice were treated with ivacaftor starting ten weeks post-MI for two consecutive weeks. Systemic ivacaftor treatment ameliorates hippocampal neuron dendritic atrophy and spine loss and attenuates hippocampus-dependent memory deficits occurring post-MI. Similarly, ivacaftor therapy mitigates MI-associated neuroinflammation (i.e., reduces higher proportions of activated microglia). Systemically, ivacaftor leads to higher frequencies of circulating Ly6C+ and Ly6Chi cells compared to vehicle-treated MI mice. Likewise, an ivacaftor-mediated augmentation of MI-associated pro-inflammatory macrophage phenotype characterized by higher CD80-positivity is observed in the MI lung. In vitro, ivacaftor does not alter LPS-induced CD80 and tumor necrosis factor alpha mRNA increases in BV2 microglial cells, while augmenting mRNA levels of these markers in mouse macrophages and differentiated human THP-1-derived macrophages. Our results suggest that ivacaftor promotes contrasting effects depending on target tissue post-MI, which may be largely dependent on its effects on different myeloid cell types.

Regional and disease specific human lung extracellular matrix composition.

Chronic lung diseases, such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), are characterized by regional extracellular matrix (ECM) remodeling which contributes to disease progression. Previous proteomic studies on whole decellularized lungs have provided detailed characterization on the impact of COPD and IPF on total lung ECM composition. However, such studies are unable to determine the differences in ECM composition between individual anatomical regions of the lung. Here, we employ a post-decellularization dissection method to compare the ECM composition of whole decellularized lungs (wECM) and specific anatomical lung regions, including alveolar-enriched ECM (aECM), airway ECM (airECM), and vasculature ECM (vECM), between non-diseased (ND), COPD, and IPF human lungs. We demonstrate, using mass spectrometry, that individual regions possess a unique ECM signature characterized primarily by differences in collagen composition and basement-membrane associated proteins, including ECM glycoproteins. We further demonstrate that both COPD and IPF lead to alterations in lung ECM composition in a region-specific manner, including enrichment of type-III collagen and fibulin in IPF aECM. Taken together, this study provides methodology for future studies, including isolation of region-specific lung biomaterials, as well as a dataset that may be applied for the identification of novel ECM targets for therapeutics.

Human alveolar hydrogels promote morphological and transcriptional differentiation in iPSC-derived alveolar type 2 epithelial cells.

Alveolar type 2 epithelial cells (AT2s) derived from human induced pluripotent stem cells (iAT2s) have rapidly contributed to our understanding of AT2 function and disease. However, while iAT2s are primarily cultured in three-dimensional (3D) Matrigel, a matrix derived from cancerous mouse tissue, it is unclear how a physiologically relevant matrix will impact iAT2s phenotype. As extracellular matrix (ECM) is recognized as a vital component in directing cellular function and differentiation, we sought to derive hydrogels from decellularized human lung alveolar-enriched ECM (aECM) to provide an ex vivo model to characterize the role of physiologically relevant ECM on iAT2 phenotype. We demonstrate aECM hydrogels retain critical in situ ECM components, including structural and basement membrane proteins. While aECM hydrogels facilitate iAT2 proliferation and alveolosphere formation, a subset of iAT2s rapidly change morphology to thin and elongated ring-like cells. This morphological change correlates with upregulation of recently described iAT2-derived transitional cell state genetic markers. As such, we demonstrate a potentially underappreciated role of physiologically relevant aECM in iAT2 differentiation.

Cystic fibrosis transmembrane regulator correction attenuates heart failure-induced lung inflammation.

Heart failure (HF) affects 64 million people worldwide. Despite advancements in prevention and therapy, quality of life remains poor for many HF patients due to associated target organ damage. Pulmonary manifestations of HF are well-established. However, difficulties in the treatment of HF patients with chronic lung phenotypes remain as the underlying patho-mechanistic links are still incompletely understood. Here, we aim to investigate the cystic fibrosis transmembrane regulator (CFTR) involvement in lung inflammation during HF, a concept that may provide new mechanism-based therapies for HF patients with pulmonary complications. In a mouse model of HF, pharmacological CFTR corrector therapy (Lumacaftor (Lum)) was applied systemically or lung-specifically for 2 weeks, and the lungs were analyzed using histology, flow cytometry, western blotting, and qPCR. Experimental HF associated with an apparent lung phenotype characterized by vascular inflammation and remodeling, pronounced tissue inflammation as evidenced by infiltration of pro-inflammatory monocytes, and a reduction of pulmonary CFTR+ cells. Moreover, the elevation of a classically-activated phenotype of non-alveolar macrophages coincided with a cell-specific reduction of CFTR expression. Pharmacological correction of CFTR with Lum mitigated the HF-induced downregulation of pulmonary CFTR expression and increased the proportion of CFTR+ cells in the lung. Lum treatment diminished the HF-associated elevation of classically-activated non-alveolar macrophages, while promoting an alternatively-activated macrophage phenotype within the lungs. Collectively, our data suggest that downregulation of CFTR in the HF lung extends to non-alveolar macrophages with consequences for tissue inflammation and vascular structure. Pharmacological CFTR correction possesses the capacity to alleviate HF-associated lung inflammation.

Restoring myocardial infarction-induced long-term memory impairment by targeting the cystic fibrosis transmembrane regulator.

BACKGROUND: Cognitive impairment is a serious comorbidity in heart failure patients, but effective therapies are lacking. We investigated the mechanisms that alter hippocampal neurons following myocardial infarction (MI). METHODS: MI was induced in male C57Bl/6 mice by left anterior descending coronary artery ligation. We utilised standard procedures to measure cystic fibrosis transmembrane regulator (CFTR) protein levels, inflammatory mediator expression, neuronal structure, and hippocampal memory. Using in vitro and in vivo approaches, we assessed the role of neuroinflammation in hippocampal neuron degradation and the therapeutic potential of CFTR correction as an intervention. FINDINGS: Hippocampal dendrite length and spine density are reduced after MI, effects that associate with decreased neuronal CFTR expression and concomitant microglia activation and inflammatory cytokine expression. Conditioned medium from lipopolysaccharide-stimulated microglia (LCM) reduces neuronal cell CFTR protein expression and the mRNA expression of the synaptic regulator post-synaptic density protein 95 (PSD-95) in vitro. Blocking CFTR activity also down-regulates PSD-95 in neurons, indicating a relationship between CFTR expression and neuronal health. Pharmacologically correcting CFTR expression in vitro rescues the LCM-mediated down-regulation of PSD-95. In vivo, pharmacologically increasing hippocampal neuron CFTR expression improves MI-associated alterations in neuronal arborisation, spine density, and memory function, with a wide therapeutic time window. INTERPRETATION: Our results indicate that CFTR therapeutics improve inflammation-induced alterations in hippocampal neuronal structure and attenuate memory dysfunction following MI. FUNDING: Knut and Alice Wallenberg Foundation [F 2015/2112]; Swedish Research Council [VR; 2017-01243]; the German Research Foundation [DFG; ME 4667/2-1]; Hjärnfonden [FO2021-0112]; The Crafoord Foundation; Åke Wibergs Stiftelse [M19-0380], NMMP 2021 [V2021-2102]; the Albert Påhlsson Research Foundation; STINT [MG19-8469], Lund University; Canadian Institutes of Health Research [PJT-153269] and a Heart and Stroke Foundation of Ontario Mid-Career Investigator Award.

Development of alginate and gelatin-based pleural and tracheal sealants.

Pleural and tracheal injuries remain significant problems, and an easy to use, effective pleural or tracheal sealant would be a significant advance. The major challenges are requirements for adherence, high strength and elasticity, dynamic durability, appropriate biodegradability, and lack of cell or systemic toxicity. We designed and evaluated two sealant materials comprised respectively of alginate methacrylate and of gelatin methacryloyl, each functionalized by conjugation with dopamine HCl. Both compounds are cross-linked into easily applied as pre-formed hydrogel patches or as in situ hydrogels formed at the wound site utilizing FDA-approved photo-initiators and oxidants. Material testing demonstrates appropriate adhesiveness, tensile strength, burst pressure, and elasticity with no significant cell toxicity in vitro assessments. Air-leak was absent after sealant application to experimentally-induced injuries in ex-vivo rat lung and tracheal models and in ex vivo pig lungs. Sustained repair of experimentally-induced pleural injury was observed for up to one month in vivo rat models and for up to 2 weeks in vivo rat tracheal injury models without obvious air leak or obvious toxicities. The alginate-based sealant worked best in a pre-formed hydrogel patch whereas the gelatin-based sealant worked best in an in situ formed hydrogel at the wound site thus providing two potential approaches. These studies provide a platform for further pre-clinical and potential clinical investigations. STATEMENT OF SIGNIFICANCE: Pneumothorax and pleural effusions resulting from trauma and a range of lung diseases and critical illnesses can result in lung collapse that can be immediately life-threatening or result in chronic leaking (bronchopleural fistula) that is currently difficult to manage. This leads to significantly increased morbidity, mortality, hospital stays, health care costs, and other complications. We have developed sealants originating from alginate and gelatin biomaterials, each functionalized by methacryloylation and by dopamine conjugation to have desired mechanical characteristics for use in pleural and tracheal injuries. The sealants are easily applied, non-cytotoxic, and perform well in vitro and in vivo model systems of lung and tracheal injuries. These initial proof of concept investigations provide a platform for further studies.

Comparative immunogenicity of decellularized wild type and alpha 1,3 galactosyltransferase knockout pig lungs.

Decellularized pig lungs recellularized with human lung cells offer a novel approach for organ transplantation. However, the potential immunogenicity of decellularized pig lungs following exposure to human tissues has not been assessed. We found that exposure of native lungs from wildtype and transgenic pigs lacking alpha (1,3)-galactosyltransferase (α-gal KO) to sera from normal healthy human volunteers demonstrated similar robust IgM and IgG immunoreactivity, comparably decreased in decellularized lungs. Similar results were observed with sera from patients who had previously undergone transcutaneous porcine aortic valve replacement (TAVR) or from patients with increased circulating anti-α-gal IgE antibodies (α-gal syndrome). Depleting anti-α-gal antibodies from the sera demonstrated both specificity of α-gal immunoreactivity and also residual immunoreactivity similar between wildtype and α-gal KO pig lungs. Exposure of human monocytes and macrophages to native wildtype lungs demonstrated greater induction of M2 phenotype than native α-gal KO pig lungs, which was less marked with decellularized lungs of either type. Overall, these results demonstrate that native wildtype and α-gal KO pig lungs provoke similar immune responses that are comparably decreased following decellularization. This provides a further platform for potential use of decellularized pig lungs in tissue engineering approaches and subsequent transplantation schemes but no obvious overall immunologic advantage of utilizing lungs obtained from α-gal KO pigs.

Improving Cerebrovascular Function to Increase Neuronal Recovery in Neurodegeneration Associated to Cardiovascular Disease.

Mounting evidence indicates that the presence of cardiovascular disease (CVD) and risk factors elevates the incidence of cognitive impairment (CI) and dementia. CVD and associated decline in cardiovascular function can impair cerebral blood flow (CBF) regulation, leading to the disruption of oxygen and nutrient supply in the brain where limited intracellular energy storage capacity critically depends on CBF to sustain proper neuronal functioning. During hypertension and acute as well as chronic CVD, cerebral hypoperfusion and impaired cerebrovascular function are often associated with neurodegeneration and can lead to CI and dementia. Currently, all forms of neurodegeneration associated to CVD lack effective treatments, which highlights the need to better understand specific mechanisms linking cerebrovascular dysfunction and CBF deficits to neurodegeneration. In this review, we discuss vascular targets that have already shown attenuation of neurodegeneration or CI associated to hypertension, heart failure (HF) and stroke by improving cerebrovascular function or CBF deficits.

Functional role of glycosaminoglycans in decellularized lung extracellular matrix.

Despite progress in use of decellularized lung scaffolds in ex vivo lung bioengineering schemes, including use of gels and other materials derived from the scaffolds, the detailed composition and functional role of extracellular matrix (ECM) proteoglycans (PGs) and their glycosaminoglycan (GAG) chains remaining in decellularized lungs, is poorly understood. Using a commonly utilized detergent-based decellularization approach in human autopsy lungs resulted in disproportionate losses of GAGs with depletion of chondroitin sulfate/dermatan sulfate (CS/DS) > heparan sulfate (HS) > hyaluronic acid (HA). Specific changes in disaccharide composition of remaining GAGs were observed with disproportionate loss of NS and NS2S for HS groups and of 4S for CS/DS groups. No significant influence of smoking history, sex, time to autopsy, or age was observed in native vs. decellularized lungs. Notably, surface plasmon resonance demonstrated that GAGs remaining in decellularized lungs were unable to bind key matrix-associated growth factors FGF2, HGF, and TGFß1. Growth of lung epithelial, pulmonary vascular, and stromal cells cultured on the surface of or embedded within gels derived from decellularized human lungs was differentially and combinatorially enhanced by replenishing specific GAGs and FGF2, HGF, and TGFß1. In summary, lung decellularization results in loss and/or dysfunction of specific GAGs or side chains significantly affecting matrix-associated growth factor binding and lung cell metabolism. GAG and matrix-associated growth factor replenishment thus needs to be incorporated into schemes for investigations utilizing gels and other materials produced from decellularized human lungs. STATEMENT OF SIGNIFICANCE: Despite progress in use of decellularized lung scaffolds in ex vivo lung bioengineering schemes, including use of gels and other materials derived from the scaffolds, the detailed composition and functional role of extracellular matrix (ECM) proteoglycans (PGs) and their glycosaminoglycan (GAG) chains remaining in decellularized lungs, is poorly understood. In the current studies, we demonstrate that glycosaminoglycans (GAGs) are significantly depleted during decellularization and those that remain are dysfunctional and unable to bind matrix-associated growth factors critical for cell growth and differentiation. Systematically repleting GAGs and matrix-associated growth factors to gels derived from decellularized human lung significantly and differentially affects cell growth. These studies highlight the importance of considering GAGs in decellularized lungs and their derivatives.

CFTR Therapeutics Normalize Cerebral Perfusion Deficits in Mouse Models of Heart Failure and Subarachnoid Hemorrhage.

Heart failure (HF) and subarachnoid hemorrhage (SAH) chronically reduce cerebral perfusion, which negatively affects clinical outcome. This work demonstrates a strong relationship between cerebral artery cystic fibrosis transmembrane conductance regulator (CFTR) expression and altered cerebrovascular reactivity in HF and SAH. In HF and SAH, CFTR corrector compounds (C18 or lumacaftor) normalize pathological alterations in cerebral artery CFTR expression, vascular reactivity, and cerebral perfusion, without affecting systemic hemodynamic parameters. This normalization correlates with reduced neuronal injury. Therefore, CFTR therapeutics have emerged as valuable clinical tools to manage cerebrovascular dysfunction, impaired cerebral perfusion, and neuronal injury.

Early Career Members at the ERS Lung Science Conference: cell-matrix interactions in lung disease and regeneration: Early career forum.

.@EarlyCareerERS looks back on #LSC2018 http://ow.ly/6hjS30jB6P9.

Avian lungs: A novel scaffold for lung bioengineering.

Allogeneic lung transplant is limited both by the shortage of available donor lungs and by the lack of suitable long-term lung assist devices to bridge patients to lung transplantation. Avian lungs have different structure and mechanics resulting in more efficient gas exchange than mammalian lungs. Decellularized avian lungs, recellularized with human lung cells, could therefore provide a powerful novel gas exchange unit for potential use in pulmonary therapeutics. To initially assess this in both small and large avian lung models, chicken (Gallus gallus domesticus) and emu (Dromaius novaehollandiae) lungs were decellularized using modifications of a detergent-based protocol, previously utilized with mammalian lungs. Light and electron microscopy, vascular and airway resistance, quantitation and gel analyses of residual DNA, and immunohistochemical and mass spectrometric analyses of remaining extracellular matrix (ECM) proteins demonstrated maintenance of lung structure, minimal residual DNA, and retention of major ECM proteins in the decellularized scaffolds. Seeding with human bronchial epithelial cells, human pulmonary vascular endothelial cells, human mesenchymal stromal cells, and human lung fibroblasts demonstrated initial cell attachment on decellularized avian lungs and growth over a 7-day period. These initial studies demonstrate that decellularized avian lungs may be a feasible approach for generating functional lung tissue for clinical therapeutics.

Preparation of Decellularized Lung Matrices for Cell Culture and Protein Analysis.

The limited available treatment options for patients with chronic lung diseases, such as fibrosis, lead to poor prognosis after diagnosis and short survival rates. An exciting new bioengineering approach utilizes de- and recellularization of lung tissue to potentially overcome donor organ shortage and immune reactions toward the received transplant. The goal of decellularization is to create a scaffold which contains the necessary framework for stability and functionality for regenerating lung tissue while removing immunomodulatory factors by removal of cells. After decellularization, the scaffold could be re-functionalized by repopulation with the patient's own stem/progenitor cells to create a fully functional organ or can be used as ex vivo models of disease. In this chapter the decellularization of lung tissue from multiple species (i.e., rodents, pigs, and humans) as well as disease states such as fibrosis is described. We discuss and describe the various quality control measures which should be used to characterize decellularized scaffolds, methods for protein analysis of the remaining scaffold, and methods for recellularization of scaffolds.